The overall aim of this proposed project is to understand the molecular mechanisms of chromatin remodeling enzymes and their role in controlling chromosomal functions. An explosion of new information on the role of chromatin dynamics in modulating gene transcription and DNA metabolism has necessitated the need to understand the molecular and regulatory mechanisms for enzymes that reversibly modify chromatin. The specific aims are directed at understanding the function of the Silent Information Regulator 2 (Sir2) family of enzymes, both at the biochemical and cellular level. Recent evidence has indicated that these proteins possess unique enzymatic activities (NAD+-dependent protein deacetylation) that are required for their chromatin silencing effects. Evolutionarily conserved, the Sir2 family has been implicated in a wide range of biological activities, including gene silencing, chromosomal stability and life-span extension via caloric restriction. We have recently discovered that Sir2 enzymes are potent histone/protein deacetylases that couple protein deacetylation to the production of a completely novel metabolite O-acetyl-ADP-ribose. New evidence suggests that generation of O-acetyl-ADP-ribose may mediate important biological functions. This metabolite may be the key to understanding the diverse biological functions of Sir2 enzymes. For instance, the generation of O-acetyl-ADP-ribose may link gene silencing in the nucleus with proper metabolic control. However, the mechanism of how Sir2 proteins accomplish this reaction and the functional significance of producing O-acetyl-ADP-ribose are not understood. To probe the biological and biochemical functions of this unique family of enzymes, this proposal provides a fully integrated approach utilizing biochemistry, proteomic methods, biochemical genomics, structure and chemistry to address how and why Sir2 enzymes catalyze this unique reaction and the production of O-acetyl-ADP-ribose. Given the vast data relating chromosomal instability and disease (e.g. cancer) with chromatin remodeling enzymes, our understanding of these molecular mechanisms may lead to the development of rationale therapeutics that inhibit Sir2 enzyme function.